1. Field of the Invention
The present invention relates to new acridone derivatives having characteristic fluorescence lifetimes that can be used as labels for attachment to and labelling of target materials. The acridone derivatives of the invention may be easily distinguished, one from the other, by virtue of their fluorescence lifetimes and they may be used in multiparameter applications. The invention also relates to assay methods utilising acridone derivatives and to a set of different fluorescent acridone lifetime dyes.
2. Description of the Prior Art
There is an increasing interest in, and demand for, fluorescent labels for use in the labelling and detection of biological materials. Fluorescent labels are generally stable, sensitive and a wide range of methods are now available for the labelling of biomolecules. Typically, the emission spectrum of a fluorescent dye is a characteristic property of the dye, the intensity of such emission being used in the detection of materials labelled with that dye. One problem with measurements of fluorescence intensity as a means of detecting and/or measuring the concentration of a fluorescent labelled biomolecule is that background fluorescence may interfere with the measurement. Thus, in order to obtain improvements in the sensitivity of fluorescence detection, it is highly desirable to improve the signal-to-noise ratio.
One means of overcoming the problem of background noise has been through the use of long wavelength dyes, for example, the cyanine dyes Cy™5 and Cy7, as disclosed in U.S. Pat. No. 5,268,486 (Waggoner et al). These dyes emit in the 600-800 nm region of the spectrum, where background fluorescence is much less of a problem. Another means of improving the signal-to-noise ratio in fluorescence measurements is in the use of time-resolved fluorescence, for example by using fluorescent labels based on lanthanide chelates, eg. Eu3+ and Tb3+ (Selvin et al, U.S. Pat. No. 5,622,821). In time-resolved fluorescent labels, the lifetime of the fluorescence emission is typically longer than that of the background fluorescence, which may therefore be gated out using appropriate instrumentation.
McGown, L. B. et al (Anal. Chem., (2000), 72, 5865-73) describe the use of a range of different dyes for multiparameter analysis in which fluorescence lifetime, rather than fluorescence wavelength, is the discriminating characteristic. Dyes from different dye classes were used to obtain lifetime resolution; however compensation was required for either mobility differences or different fluorescence signal intensities. The method has been refined by Sauer, M. et al (J. Fluorescence, (1993), 3(3), 131-139) who employed a series of rhodamine-based fluors having a range of fluorescent lifetimes but which all absorb and emit at similar wavelengths, thus avoiding having to change the excitation source and emission filters.
The acridone chromophore is highly fluorescent and has been used for labelling biological molecules and subsequent detection by conventional fluorescence emission spectroscopy. For example, Faller, T. et al (J. Chem. Soc. Chem. Comm., (1997), 1529-30) describe the preparation of a succinimidyl ester derivative of acridone and its use in labelling peptides for subsequent analysis by mass-spectroscopy. U.S. Pat. No. 5,472,582 (Jackson) describes the use of the fluorescent label, 2-aminoacridone, for labelling and detecting carbohydrates in a mixture, following electrophoretic separation.
Val'kova, G. et al (Dokl. Akad. Nauk. SSR, (1978), 240(4), 884-7) have measured the fluorescence lifetimes of several acridone derivatives, however, to date, there appear to be no reports relating to the use of acridones as lifetime dyes suitable for labelling and the detection of biological materials.